A View on Cathepsin C as a Target for Therapy in AAV

The cathepsins, members of the large family of papain-like cysteine peptidases, have become the subject of intense research because of their increasingly well-defined roles in immune responses, both in homeostatic conditions and in autoimmunity and the host response to tumors.^1,2 Of particular note is the role of the endosomal/lysosomal aminopeptidase cathepsin C (CatC) in coordinating activation of serine proteases released from azurophilic granules of stimulated neutrophils.^1–3 These proteases, which include neutrophil elastase, proteinase 3 (PR3), and neutrophil serine protease 4 (NSP4), are an important defense against bacterial infection and collateral tissue injury.^1,2 They are thought to be responsible for the hyperacute inflammation in the lung injury mediated by severe acute respiratory syndrome coronavirus 2.^3,4 More generally, in chronic inflammatory lung disease, protein concentrations and enzymatic activity of CatC in lung secretions correlate with the number of neutrophils in bronchoalveolar lavage fluid.

Active CatC could be a biomarker for neutrophilic inflammation and neoplastic disease^5,6 and a potential therapeutic target because effective CatC inhibition would prevent activation and tissue damage from multiple NSPs.⁴ This is a particularly tantalizing prospect in ANCA-associated vasculitis (AAV) because PR3 is the principal target of ANCAs in the granulomatosis with polyangiitis form of AAV. Thus, in addition to its nonspecific effect on neutrophil-mediated injury, CatC inhibition might limit autoantibody-mediated injury by reducing PR3 availability.

In this issue, Jerke et al.⁷ use a rare “experiment of nature” to investigate involvement of CatC in microvascular injury, as in AAV. Papillon–Lefèvre syndrome (PLS) is an autosomal recessive disease, first described in 1924, that is caused by loss of function of CTCS, the gene coding for CatC.⁸ Severe epidermal disease in PLS has been attributed to neutrophil dysfunction, most commonly palmoplantar keratoderma characterized by scaling, fissures, and frequent pyogenic infections, typically with Staphylococci. Periodontitis, also common, can cause premature loss of teeth. Seren and colleagues have reported that, despite similar abundance of Pr3m on the surface of quiescent neutrophils from healthy donors and those with PLS, marked differences emerged after neutrophil activation: Pr3m increased in normal neutrophils, but not in neutrophils from individuals with PLS.³ They concluded that CatC deficiency impairs PR3 maturation, reducing surface expression of Pr3m, and speculated about benefits of inhibiting CatC in AAV. In this study, they now address this question further.

Neutrophils and monocytes were purified from healthy donors and two patients with PLS. Cellfree supernatants were generated and tested for their ability to cause NSP-mediated endothelial cell injury in the presence or absence of a pharmacologic CatC inhibitor. The researchers confirmed PLS neutrophils and monocytes expressed less PR3, neutrophil elastase, and CatG than normal neutrophils, and showed significantly less proteolytic activity in the lysates of PLS monocyte-enriched populations. Not surprisingly, serum from a patient with AAV with anti-PR3 ANCA did not yield a characteristic cytoplasmic ANCA pattern when incubated with PLS neutrophils.

In contrast to the previous study,³ PLS neutrophils’ expression of Pr3m was reduced compared with healthy neutrophils, and did not show the normal bimodal pattern of distinct populations of CD177^neg/mPR3^lo and CD177^pos/mPR3^hi cells. Pr3m expression was also reduced in PLS monocytes. Differences in background levels of cell activation in the two studies could explain reduced Pr3m expression in neutrophils and monocytes. The authors addressed whether supernatants from activated PLS neutrophils with reduced NSPs levels, including PR3, damage endothelium less using human umbilical vein endothelial cells and then, more relevantly, glomerular microvascular endothelial cells—the target cell in renal AAV or focal necrotizing (and crescentic) GN. Incubation with cellfree supernatant from healthy neutrophils significantly injured glomerular microvascular endothelial cells. Endothelial injury was associated with enzymatically active, cell-associated PR3. Supernatants from PLS neutrophils caused no injury or detectable cell-associated PR3. These results contribute to a long-standing discussion of whether PR3 on endothelial cells is available for anti-PR3 ANCA binding—however, these data require replication and confirmation.

Importantly, the authors replicated the PLS neutrophil results pharmacologically by inhibiting CatC in healthy neutrophils incubated with the small-molecule CatC inhibitor BI-9740, while injury was not reduced using supernatants from neutrophils incubated with corticosteroids. The experiments provide a rationale for further studies of CatC inhibition as potential therapy for AAV, but will require safety assessment in vitro and confirmation in an in vivo model. CatC inhibition could cause immunodeficiency due to inhibition of neutrophil differentiation, because PR3 is identical to myeloblastin, which promotes growth and differentiation in promyelocytic cells.⁹ However, this seems unlikely because neither the patients with PLS presented here nor those described in the literature exhibited obvious generalized immunodeficiency. CatC appears to control NSP activation, but not immune cell differentiation. Another neutrophil defect in PLS is the inability to produce neutrophil extracellular traps, which have powerful pathogen immobilizing and antimicrobial activities and have been implicated in the pathogenesis of AAV, independent of ANCA specificity or titer.¹⁰ Therefore, even short-term therapeutic inhibition could lead to neutrophil dysfunction and periodontitis, as seen in other lysosomal disorders.¹¹ Finally, in PLS, lack of NSP activation leads to autophagic dysfunction and the absence of NSP proteolytic activity, resulting in failure to degrade gram-negative and gram-positive bacteria and consequent inflammation.

These investigations confirm that CatC is a central processing enzyme for NSP activation and for the function of the organelle in which they are contained—the lysosome. Lysosomal function is dependent on two classes of proteins: membrane (glyco-)proteins and enzymes (predominantly soluble, lysosomal hydrolases).¹² In the past, lysosomes were viewed as compartments that house waste-digesting enzymes with mutations of their constituent proteins resulting in lysosomal storage diseases. More recently, they have been recognized as part of a broader cellular compartment, the lysosomal-endosomal system, which has a central role in phagocytosis and pathogen elimination and links multiple processes involved in autoimmunity, including autophagy and antigen presentation. Lysosomal cathepsins potently modulate the (innate) immune response.¹² For example, CatC cleaves toll-like receptors (TLRs), such as TLR9 and IL-1β , potentially interfering with recruiting neutrophils and monocytes to sites of injury.¹³ Thus, cathepsins are important for pathogen detection and signaling, and for processing and presenting antigens to T lymphocytes.

Preclinical studies will be the next step in examining systemic CatC inhibition to treat AAV. However, choosing an appropriate in vivo model is challenging. Widely used models of focal necrotizing GN could test the generalized anti-inflammatory effects of CatC inhibition, but may not specifically determine whether CatC inhibition of PR3 is sufficiently powerful in vivo to reduce neutrophil activation (and thus injury) in AAV induced by autoantibodies to PR3. The authors’ in vitro experiments suggest this may be so by demonstrating PR3 ANCAs were unable to activate CatC-deficient neutrophils, whereas anti–myeloperoxidase-ANCAs (anti–MPO-ANCAs) retained this ability. An anti-PR3–mediated murine model of AAV would be appropriate, but none is currently available. Nevertheless, well-established mouse models of anti-MPO–mediated injury could test whether potent, but nonspecific, CatC inhibition reduces in vivo NSP activity and attenuates injury in focal necrotizing GN.

Effects of CatC inhibition are being investigated in preclinical models of other chronic inflammatory diseases. For AAV, there is hope that downstream effects on inflammation may be important, irrespective of whether autoimmunity to PR3 or MPO are the initiating pathogenic events. CatC inhibition also reduces formation of neutrophil extracellular traps, which are abundant in glomerular capillaries in ANCA-associated focal necrotizing GN and are thought to contribute to injury, regardless of autoantibody specificity.¹⁰ Ultimately, whether CatC inhibition will be a valuable treatment for AAV more generally will be critically dependent on carefully dissecting the molecular mechanisms in both settings.

Disclosures

R. Kain reports having consultancy agreements with AstraZeneca, Janssen Cilag, MEDahead, and Takeda Pharma; receiving honoraria from AstraZeneca, Janssen Cilag, and Takeda Pharma; and having other interests in, or relationships with, the Austrian Society Pathology (as president), Horizon Europe (European Commission), the Renal Pathology Society (RPS; as councilor), and RPS International Committee. The remaining author has nothing to disclose.

Funding

None.

Author Contributions

R. Kain wrote the original draft; R. Kain and M. Nackenhorst conceptualized the study; and M. Nackenhorst reviewed and edited the manuscript.